CHARACTERIZATION OF THE COATED VESICLE PROTON PUMP

涂层囊泡质子泵的表征

• 批准号: 3285551
• 负责人: MICHAEL D FORGAC
• 金额: $ 16.97万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-08-30 至 1993-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3285551
• 关键词: Golgi apparatus; adenosine triphosphate; adenosinetriphosphatase; clathrin; enzyme reconstitution; enzyme structure; enzyme substrate complex; immunochemistry; laboratory mouse; laboratory rabbit; laboratory rat; lysosomes; membrane model; membrane proteins; membrane reconstitution /synthesis; membrane structure; monoclonal antibody; phospholipids; receptor mediated endocytosis

The objectives of this research are to determine the structure, ion transport properties and intracellular distribution of the ATP- dependent proton pump of clathrin-coated vesicles. This pump appears to play s crucial role in intracellular membrane traffic by providing the acidic environment required for ligand-receptor dissociation and receptor recycling following receptor-mediated endocytosis. In addition, the coated vesicle pump represents a new class of ATP-driven proton pumps which acidify a variety of intracellular compartments, including endosomes, lysosomes, Golgi and the vacuoles of plants and lower eukaryotes. Our work will focus on further structural characterization of the coated vesicle proton pump and on determination of its relationship to the proton pumps of other intracellular organelles. We have succeeded in isolation of the coated vesicle (H+)-ATPase and in reconstitution of this pump into artificial lipid vesicles. The purified enzyme contains nine polypeptides of molecular weight 17,000-100,000. On the basis of labeling studies we have suggested that the 73,000 dalton subunit is involved in ATP hydrolysis and the 17,000 dalton subunit forms part of a DCCD-inhibitable proton channel. Further studies are planned to characterize the reactive sites on these polypeptides. Subunit stoichiometry and proximity will be measured by amino acid analysis and covalent crosslinking, respectively. Subunits will be tested for glycosylation or peripheral membrane association, and the disposition of each subunit with respect to the membrane will be determined by labeling with membrane impermeant and hydrophobic reagents and by proteolysis. The ion transport properties of the intact complex and the isolated 17,080 dalton subunit will also be investigated. We have also succeeded in isolation of a series of monoclonal antibodies which recognize the native, detergent-solubilized (H+)- ATPase and immuneprecipitate this enzyme in an active form. These antibodies will be used to test the immunological relationship between the various intracellular proton pumps using both immunocytochemical techniques and by immuneprecipitation of crossreactive species from isolated organelles. These antibodies will also be used to probe for the presence of this pump in the plasma membrane.

本研究的目的是确定结构，离子 ATP的转运特性和细胞内分布- 依赖于网格蛋白包被囊泡的质子泵。 这个泵 似乎在细胞内膜交通中发挥着关键作用 通过提供配体-受体所需的酸性环境 受体介导的解离和受体再循环 内吞作用 此外，涂层囊泡泵代表了一种新的 一类ATP驱动的质子泵，可酸化各种 细胞内区室，包括内体、溶酶体、高尔基体 以及植物和低等真核生物的液泡。 我们的工作将集中在进一步的结构表征， 包被囊泡质子泵及其相互关系的测定 其他细胞器的质子泵。 我们有 成功地分离了包被囊泡（H+）-ATP酶， 将该泵重构成人工脂质囊泡。 的 纯化的酶含有9种分子量的多肽 17,000-100,000. 根据标签研究，我们建议 73，000道尔顿亚基参与ATP水解，并且 17，000道尔顿亚基形成了一个DCCD可转换质子的一部分 频道 计划进行进一步的研究以表征反应性 这些多肽上的位点。 亚基化学计量和邻近性 将通过氨基酸分析和共价交联来测量， 分别 将检测亚单位的糖基化或 外周膜联合，以及每一个的处置 相对于膜的亚基将通过标记 用膜不渗透和疏水试剂， 蛋白水解 完整络合物的离子输运性质 并且还将研究分离的17，080道尔顿亚基。 我们还成功地分离了一系列单克隆抗体， 识别天然的、洗涤剂溶解的（H+）- ATP酶和免疫沉淀这种酶的活性形式。 这些 抗体将用于测试免疫学关系 在各种细胞内质子泵之间， 免疫细胞化学技术和免疫沉淀 来自孤立细胞器的交叉反应物种。 这些抗体 也将用于探测该泵在 质膜